Production of deoxyribonucleoside triphosphate (dNTP) precursors for DNA synthesis is critical for cell proliferation and genomic integrity. Two biosynthetic pathways contribute to cellular dNTP pools: de novo synthesis and deoxyribonucleoside salvage. This application focuses on deoxycytidine kinase (DCK), a key enzyme in the salvage pathway. DCK has unique properties: it provides cells with all 4 dNTPs and is essential for the activation of nucleoside analog drugs used in cancer. We hypothesize that the enzymatic activity of DCK can be imaged by Positron Emission Tomography (PET) and that PET assays that measure DCK may allow stratification of cancer patients for treatment with nucleoside analog drugs. To test these hypotheses we propose a multidisciplinary project that combines genetic, molecular biology, biochemical and molecular imaging approaches to investigate the biological function of DCK and to develop PET probes to monitor its activity in vivo. This work leverages tools and reagents identified in the current ICMIC cycle; we developed [ [18] F]-1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine ([ [18]F] FAC), a new DCK substrate PET probe, using a new approach to identify potential imaging agents. The team of proposed investigators has combined excellence in cancer biology, molecular biology, biochemistry, radiochemistry, preclinical and clinical molecular imaging. The strength of this collaboration is demonstrated by the fact that in the last 24 months we have taken a series of PET probes from extensive in vitro selection and evaluation to in vivo PET investigations in mice, and then to patients. This project will use all three ICMIC Specialized Resources. In Specific Aim 1 we will develop a novel experimental model that allows conditional inactivation of the DCK gene in mice. The DCK deficient mice will be used to validate the tissue retention of [ [18]F] FAC as an accurate non-invasive measurement of DCK activity in vivo and to interpret the results of PET assays using DCK-specific probes. In Specific Aim 2 we will design, synthesize and evaluate optimized [ [18]F] FAC probes. Specific Aim 3 proposes the clinical translation of DCK-specific probes and the development of new molecular imaging approaches to predict tumor responses to DCK-dependent drugs.